Diagnostic testing process

ABSTRACT

A method and apparatus for use in a flow through assay process is disclosed. The method is characterised by a “pre-incubation step” in which the sample which is to be analysed (typically for the presence of a particular protein), and a detection analyte (typically one or more antibodies bound to colloidal gold or a fluorescent tag) which is known to bind to the particular protein may bind together for a desired period of time. This pre-incubation step occurs before the mixture of sample and detection analyte come into contact with a capture analyte bound to a membrane. The provision of the pre-incubation step has the effect of both improving the sensitivity of the assay and reducing the volume of sample required for an assay. An apparatus for carrying out the method is disclosed defining a pre-incubation chamber for receiving the sample and detection analyte having a base defined by a membrane and a second membrane to which a capture analyte is bound. In one version the pre-incubation chamber is supported above the second membrane in one position but can be pushed into contact with the membrane carrying the capture analyte thus permitting fluid transfer from the incubation chamber through the capture membrane. In another version the membrane at the base of the incubation chamber is hydrophobic and its underside contacts the capture membrane and when a wetting agent is applied to the contents of the pre-incubation chamber fluid transfer occurs.

FIELD OF THE INVENTION

This invention relates to a diagnostic testing process and in particularto an apparatus for use in carrying out an assay process and to a methodof carrying out an assay process using that apparatus.

BACKGROUND OF THE INVENTION

Background Art

Lateral flow and flow-through technology have been used for diagnosticassays for almost twenty years. Lateral flow technology is currentlydominant because lateral flow devices are easy to produce and the assaycan be performed in a simple 2-step process that can be adapted forwhole blood separation. This results in a simple device that can be usedin the field as a rapid point-of-care diagnostic (Cole et al 1996Tuberc. Lung. Dis. 77:363-368). However, multiple disease diagnosisusing lateral flow technology is very difficult because of differencesin lateral diffusion between samples and variation in flow rates betweenbatches of the partitioning membrane. This means that antigen orantibody signal strengths may vary both within tests and between batchesof tests, resulting in inconsistent results.

Existing flow-through diagnostic tests can be completed in less than twominutes compared with typical times of five to fifteen minutes forlateral flow tests. This advantage in speed however, is often at theexpense of sensitivity. A further disadvantage is that higher volumes ofsample are required to achieve the same sensitivity as lateral flow.This may be problematic in some situations. For example, the diagnosisof analytes (reagents) in whole blood requires the separation of plasmafrom whole blood cells. The higher volumes of whole blood required forthis would quickly block the membranes in the flow-through format.

The basic principal of flow-through assays is well established. Thetests are designed to determine the existence of, and in some cases, thequantity of, a predetermined analyte/reagent in a sample. Often thereagent will be a protein but other reagents can be tested for. If theassay is to test for the existence of a particular disease in a patient,the patient's body fluids may be tested for an antibody or other proteinproduced by the patient in response to the infection, or for a proteinwhich is expressed by the bacterium or viral agent or the like causingthe disease. In a typical flow through assay a liquid sample which isbelieved to contain the reagent is sucked into an absorbent pad via amembrane to which is bound a capture analyte which is known to bind tothe reagent. The membrane is then typically washed with a buffer and aliquid containing a detection analyte which also binds to the reagentand which includes a tracer or marker which is detectable, is applied tothe membrane. The detection analyte binds to the immobilised reagentbound to the membrane and can be seen or otherwise detected to indicatethe presence of the reagent.

U.S. Pat. No. 4,246,339 discloses a test device for assaying liquidsamples for the presence of a predetermined reagent. The device includestelescoping top and bottom members defining a liquid reservoirtherebetween and resilient means for biasing the members in the openposition. The top member defines a series of test wells each of whichhas a base defined by a microporous membrane with a capture analyteimmobilised on the membrane surface. Absorbent means are located in thebottom member, spaced from the membrane in the open position but incontact therewith in the closed position. U.S. Pat. No. 4,246,339discloses adding the test serum diluted with a buffer to a test well,and incubating the device at room temperature for ten minutes prior todepressing the cassette to the closed position to pass the samplethrough the membranes into the absorbent material, When the membranesare dry, the membrane is washed and then covered with a solutioncontaining a detection analyte which binds to the immobilised reagentfollowed by a subsequent step in which a stain is applied.

It will be appreciated that the process described in U.S. Pat. No.4,246,339, is a somewhat long drawn out, time consuming and tediousprocess and also lacks sensitivity.

A more recent flow through device is described in U.S. Pat. No.5,185,127 which discloses an assay device including a filter stack andan enclosure having a base portion and a lid. The filter stack has ahydrophilic membrane having a capture analyte thereon, referred to inU.S. Pat. No. 5,185,127 as a binder. A hydrophobic membrane is locatedunder the hydrophilic membrane and a pad of absorbent material islocated under the hydrophobic membrane. The lid includes an upwardlyextending rib which defines a recess having an insert therein. In use, asample containing the reagent (referred to in U.S. Pat. No. 5,185,127 asthe analyte) is placed in the well of the assay device at which time thereagent/analyte binds to the capture analyte/binder. Flow of the assaysolution however, does not take place because the aqueous solution doesnot wet the hydrophobic membrane placed under the hydrophilic membranein the filter stack. Thus as much time is necessary to complete thebinding of the detection analyte to the reagent is allowed. When bindingis judged to be complete, flow may be initiated by adding a wettingagent which wets the hydrophobic membrane. After which time the aqueousliquid flows into pad of absorbent material. The membrane may then bewashed and treated with a detection analyte/tracer which may be anantibody which specifically binds to the analyte, the antibody having alabel covertly conjugated thereto. Again the sensitivity of U.S. Pat.No. 5,185,127 is lacking and is not equivalent to that obtainable inlateral flow or ELISA formats.

General Information.

As used herein the terms “derived from” or “derivative” shall be takento indicate that a specified integer may be obtained from a particularsource albeit not necessarily directly from that source.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

The embodiments of the invention described herein with respect to anysingle embodiment and, in particular, with respect to an apparatus or amethod of assaying shall be taken to apply mutatis mutandis to any otherembodiment of the invention described herein.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificexamples described herein. Functionally-equivalent products,compositions and methods are clearly within the scope of the invention,as described herein.

The present invention is performed without undue experimentation using,unless otherwise indicated, conventional techniques ofimmunocytochemistry such as for example immunogold labeling andproteomics. Such procedures are described, for example, in the followingtexts that are incorporated by reference:

-   -   Colloidal Gold—A New Perspective For Cytochemical Marking,        Beesley J (1989), Royal Microscopical Society Handbook No 17.        Oxford Science Publications. Oxford University Press.        (Paperback);    -   An Introduction To Immunocytochemistry Current techniques and        problems, Polak J and Van Noorden 5 (1984) Royal Microscopical        Society Handbook No 11. Oxford Science Publications. Oxford        University Press. (Paperback);    -   Immunocytochemistry—Modern Methods and Applications, Polak J and        Van Noorden 5 (1986) (2nd ed), Butterworth Heinemann Oxford.        (Hardback);    -   Techniques in Immunocytochemistry, Bullock G and Petrusz P        (1982-1989) (4 volumes) Academic Press. Paperback); and    -   Colloidal Gold—Principles, Methods and Applications, Hayat M,        (1989-1990) (3 volumes), Academic Press. (Hardback).

All the references cited in this application are specificallyincorporated by reference herein.

Because the prior art is not consistent in its terminology, for theavoidance of doubt and for the purpose of clarity, the following termsused in the specification below, are defined as follows. The term“reagent” or “target analyte” is used to refer to a macromolecule (eg.protein or enzyme) or fragment thereof, or the like which is to bedetected by an assay. The term “capture analyte” is used to refer to a“capture” compound which is bound to a membrane and to which the reagentwill bind. The term “detection analyte” is used to refer to an analytewhich comprises a “detection” compound which will also bind to thereagent and also a “detectable” element. The detectable element istypically visually detected whether under visible light, orfluorescence.

SUMMARY OF THE INVENTION

In a first broad aspect, the present invention provides an apparatus andmethod for use in an assay process which is characterised by providing a“pre-incubation step” in which a reagent and detection analyte may bindtogether, which has the effect of both improving the sensitivity of theassay and reducing the volume of sample required for an assay prior toreaction of the sample/analyte complex with a reaction membrane to whichone or more ligands are bound.

In one embodiment, the detection analyte is a multi-detection analytecomprising more than one detection compound bound to a detectableelement.

Thus, in one aspect of the present invention there is provided anapparatus for use in an assay process comprising:

-   -   a first member comprising a first, porous, reaction membrane to        which is bound a capture analyte for binding to a reagent to be        detected, the member having an upper surface and a lower        surface;    -   a second member being a body of absorbent material such as        tissue paper or the like disposed below and touching the lower        surface of the first member;    -   a chamber spaced above the first member said chamber having side        walls, and a base defined by a second membrane; and    -   means for supporting the chamber above the first member in two        positions, a first position in which the membrane is spaced a        sufficient distance from the first member so as to not permit        fluid transfer from the chamber to the body of absorbent        material, and a second position in which the membrane is in        contact with the first member thus permitting fluid transfer        from the chamber through the first and second membranes to the        body of absorbent material.

In a related aspect the present invention provides a method for assayingfor the presence of a pre-determined reagent using an apparatus of thepresent invention comprising the steps of:

-   -   a) placing a sample to be assayed and a detection analyte in the        chamber, with the chamber disposed in the first position;    -   b) allowing a sufficient period of time to pass for the        detection analyte to bind to the reagent, if present;    -   c) depressing the chamber to the second position to contact the        base of the chamber with the first porous membrane;    -   d) allowing the sample to flow through the first and second        membranes to allow the reagent, if present to bind to the        capture analyte carried on the first membrane,        preferably wherein the detection analyte is a multi-detection        anlayte.

As used herein “multi-detection analyte” refers to a detection analytewhich comprises more than one detection compound bound to a detectableelement.

The inventors have surprisingly found that a multi-detection analyte hasa number of advantages according to the present invention including, anyof the following, for example,

-   -   multiple-detection compounds can be bound to detectable elements        at optimum binding efficiency,    -   detection analytes produced with multiple detection compounds        have a similar size and physical characteristics for each of the        detection compounds,    -   the ratio of detection compounds on the detectable element can        be altered without affecting relative binding profiles of the        detectable element,    -   quantative comparisons can be performed, and    -   the detectable element has a reduced capacity for non-specific        binding.

Accordingly, in a preferred embodiment the invention provides a methodfor assaying for the presence of at least one pre-determined reagentcomprising the steps of:

-   -   a) providing a first porous membrane to which capture analytes        for binding to the at least one reagent have been bound;    -   b) placing a sample to be assayed and a multi-detection analyte        in a chamber having a base defined by a second porous membrane;    -   c) allowing a sufficient period of time to pass for the        multi-detection analyte to bind to the at least one reagent, if        present;    -   d) contacting the base of the chamber with the first porous        membrane; and    -   e) causing the sample to flow through the membranes to allow the        reagent to bind to the capture analyte carried on the first        membrane.

Preferably, the detection compound is an antigen, antibody, or ligand.

Antibodies, antigens and ligands can be used as detection compounds asthey have binding sites capable of specifically binding to the proteinsof interest or fragments or epitopes thereof in preference to othermolecules.

Antibodies are obtainable from a commercial source, Or alternatively,produced by conventional means. Commercial sources will be well known tothose skilled in the art.

As used herein the term “specifically bind” with reference to acomplex-forming agent such as an antibody, refers to the agentpreferentially associating with a target analyte (eg. protein ofinterest). With respect to antibodies in particular, it is recognisedthat a certain degree of non-specific interaction may occur between amolecule and a non-target protein. Nevertheless, specific binding may bedistinguished as mediated through specific recognition of the antigen.

In alternate embodiments, the detection compound (antibody or ligand) islinked to an element to detect target analyte in a sample by directly orindirectly labelling the antibody or ligand, e.g with radioactive,fluorescent or enzyme labels, such as horseradish peroxidase) so thatthey can be detected using techniques well known in the art. Directlylabelled analytes have a label (detectable element) associated with orcoupled to the detection compound. Indirectly labelled detectionelements may be capable of binding to a labelled species (eg. a labelledantibody capable of binding to the developing agent) or may act on afurther species to produce a detectable result.

Detectable elements can be conjugated to antibodies (or other ligands)and include macromolecular colloidal particles (e.g. colloidal goldparticles) or particulate material such as latex beads that arecoloured, magnetic or paramagnetic, and biologically or chemicallyactive agents that can directly or indirectly cause detectable signalsto be visually observed, electronically detected or otherwise recorded.These molecules may be enzymes which catalyse reactions that develop orchange colours or cause changes in electrical properties, for example.They may be molecularly excitable, such that electronic transitionsbetween energy states result in characteristic spectral absorptions oremissions. They may include chemical entities used in conjunction withbiosensors. In one embodiment, the detection compound (antibody) isconjugated to a colloidal gold particle. Binding of the conjugatedantibody to the sample protein produces a visual signal that can bedetected by eye or read electronically to give a quantitative result.Biotin/avidin or biotin/streptavidin and alkaline phosphatase detectionsystems may also be employed.

Other labels are known to those skilled in the art.

Preferably, the detection element is selected from the group comprisingcolloidal gold particle, latex bead, coloured dye and colloidal carbon.

In one embodiment, the detectable element is colloidal gold. Preferablythe colloidal gold provides uniform reflectance properties.

In an alternate embodiment, the detectable element is a latex beadlinked to a coloured dye.

In another embodiment, the detectable element is colloidal carbon.

Preferably, a positive result is where the signal from a “test” samplein the assay is significantly higher or lower than a sample from acontrol sample.

Thus, the present invention provides a chamber which may serve as apre-incubation chamber in which a pre-incubation step can occur wherethe sample and multi-detection analyte combine, which improves thesensitivity of the test and reduces the volume of sample required forthe assay. It has been found that the pre-incubation step increases thetest sensitivity for a typical existing flow-through apparatus byapproximately ten times to equivalent levels of sensitivity comparedwith lateral flow technology, while still allowing the assay to becompleted in around two minutes compared to 10 minutes for lateral flowformats.

In one embodiment the sample is selected from or is derived from thegroup comprising: agricultural product, microbial product, andbiological product.

In one embodiment, the agricultural product is selected from the groupcomprising a seed, grain or plant extract.

In one embodiment, the sample contains particulate materials selectedfrom the group comprising grain extract, cell extract and microbialextract.

In an alternate embodiment, the biological sample is a bodily fluid ortissue sample selected from the group comprising: blood, serum, sputum,and lung.

Other samples are not excluded.

Standard methods can be used to obtain, prepare and/or store samples foruse in the present invention. For example, in one embodiment anadsorbent swab comprising a cotton matrix or similar material can beused to probe or surface or medium that may contain a target analyte. Inone embodiment, the adsorbent swab is washed to remove the targetanalyte. Preferably, the target analyte and/or the particulate materialcomprising the target analyte is then solubilised.

Accordingly, in one embodiment a ground wheat head suspension can besolubilised, and then mixed and pre-incubated in the chamber with amulti-detection analyte comprising a primary antibody againstalpha-amylase linked to a colloidal gold particle and a controlsecondary antibody also linked to the colloidal gold particle. Thecontents of the chamber are then allowed to flow through to the firstmembrane containing a capture analyte in the form of an immobilisedanti-amylase antibody, or anti-control antibody and antibody/goldcomplexes will bind to the immobilised antibody forming a detectablesignal. The signal can be detected by the removal of the pre-incubationunit and washing of the reaction membrane with buffer.

In another embodiment the invention can also be used for detectingreagents in whole blood since whole red blood cells can be removed inthe pre-incubation chamber and the plasma allowed to flow-through to thereaction membrane containing a bound capture analyte. In this format,the base membrane defined at the base of the pre-incubation chamber willtypically be a membrane which has the correct pore size to retain thered blood cells and allow the plasma to pass through on contact with thefirst membrane. Similarly particulate samples containing grain extracts,cell or microbial extracts can be analysed with this flow-through formatsince particulate matter can be removed in the pre-incubation chamberand therefore cannot block the reaction area on the upper surface of thereaction membrane.

The apparatus can also be used for detecting analytes in body fluidsother than blood, such as plasma, sera, urine, saliva and sputum. Inthis case, the sample can be retained in the pre-incubation chamber byuse of a hydrophobic membrane. To obtain efficient flow throughcapillary action to the second member when the pre-incubation chamber islowered, the reaction membrane is pre-wet with a wetting agentcontaining a detergent or the reaction membrane is blocked with ahygroscopic solution such as sucrose, trehalose, fructose, oralternatively, glycerol.

This changes the characteristics of the reaction membrane from anon-hygroscopic to a hygroscopic membrane allowing the sample to flowthrough to the second member upon contact of the membrane at the base ofthe pre-incubation chamber with the reaction membrane.

In a yet further embodiment, if a hydrophobic membrane is used as thebase of the pre-incubation chamber, the apparatus may be used with thehydrophobic membrane and reaction membrane in contact, with the operatoradding a wetting agent to the sample to cause flowthrough, when desired.

Thus, in a related aspect, there is provided an apparatus for use in anassay process comprising a housing including:

-   -   a first member comprising a first, porous, membrane to which is        bound a capture analyte for binding to a reagent to be detected,        the member having an upper surface and a lower surface;    -   a second member being a body of absorbent material such as        tissue paper or the like disposed below and touching the lower        surface of the first member;    -   a chamber located above the first member said chamber having        side walls, and a base including a second, hydrophobic,        membrane, having an upper and a lower surface, the        pre-incubation chamber being supported above the first member        with the lower surface of the hydrophobic membrane in contact        with the upper surface of the first member.

The pre-incubation chamber can also be used to remove analytes that mayinterfere with the assay, such as human anti-mouse antibodies (HAMAS),in solution or by binding anti-analyte antibodies to the surface of thechamber. The chamber can also be used to extract the analyte of interestfrom an absorbent surface such as a swab, which has been taken from thethroat of a patient, by swirling the swab in an extraction solution inthe chamber. The pre-incubation chamber may be part of a pre-filter unitwhich acts also to pre-filter the sample prior to contact with the uppersurface of the first member.

Examples of assays that can be peformed by this method where tworeaction steps are involved (the incubation of the analyte with thelabeled anti-analyte followed by the binding of this complex to asolid-phase anti-analyte), are:

-   -   Direct antigen assay        -   1. Ag*(analyte)+Ab*₁(anti-Ag)-label        -   2. Solid phase-Ab₂(anti-Ag)+Ag/Ab₁(anti-Ag)-label complex    -   Direct antibody assay (i)        -   1. Ab₁(analyte=anti-Ag)+Ab₂(anti-Ab₁)-label        -   2. Solid phase-Ag+Ab₁(anti-Ag)/Ab₂(anti-Ab₁)-label complex    -   Direct antibody assay (ii)        -   1. Ab₁(analyte=anti-Ag)+Ab₂(anti-Ab₁)-label        -   2.            Solid-phase-Ab₃(anti-Ag)/Ag+Ab₁(anti-Ag)/Ab₂(anti-Ab₁)-label            complex    -   Indirect antigen assay        -   1. Ag (analyte)+Ab₁(anti-Ag)+Ab₂(anti-Ab₁)-label        -   2.            Solid-phase-Ab₃(anti-Ag)+Ag/Ab₁(anti-Ag)/Ab₂(anti-Ab₁)-label            complex    -   Indirect antibody assay (i)        -   1. Ab₁(analyte=anti-Ag)+Ab₂(anti-Ab₁)+Ab₃(anti-Ab₂)-label        -   2. Solid phase            Ag+Ab₁(anti-Ag)/Ab₂(anti-Ab₁)/Ab₃(anti-Ab₂)-label complex    -   Indirect antibody assay (ii)        -   1. Ab₁(analyte=anti-Ag)+Ab₂(anti-Ab₁)+Ab₃(anti-Ab₂)-label        -   2. Solid phase            Ab₄(anti-Ag)/Ag+Ab₁(anti-Ag)/Ab₂(anti-Ab₁)/Ab₃(anti-Ab₂)-label            complex    -   *Ag indicates antigen    -   *Ab indicates antibody

Alternate types of assays are not excluded.

A piezoelectric driven printer may be used to dispense precise amountsof multiple disease ligands such as antigens or antibodies or an analyteas a micro array onto a reaction membrane for use in the apparatus ofthe first aspect of the present invention. The ligands or analytes maybe dispensed in particular patterns, e.g. letters for ease ofrecognition of results. Typically, 100 pl of fluid reagent (1 drop), ormultiples thereof, is dispensed, but this will vary depending on theapplication. The resultant size of the spot on the membrane is about 55microns or more in diameter subject to fluid diffusion on the membrane,but again this will vary depending on the application. It is possible todispense droplets with diameters of 5-10 microns, and hence lowervolumes of fluid reagent (for example, 1-10 pl) can be applied. Usingprecise quantitative printing of micro arrays of antibodies, antigens,or other analytes means that tests using precise quantities of thesereagents can be produced for multi disease diagnosis of a single sample.This array technology can be applied to tests for drugs or other markersacross all diagnostic fields.

Alternatively, an adult/neonatal syringe pump 1235 from ATOM MedicalCorporation, Japan, typically used to administrator small quantities ofintravenous liquids through a catheter to hospital patients can beadapted to apply single or multiple lines of a capture analyte to thefirst membrane eg nitrocellulose.

In one preferred embodiment, ligands for detecting tuberculosis, HIV,hepatitis, syphilis and malaria antibodies may be deposited onto areaction membrane. This would allow the simultaneous diagnosis oftuberculosis, HIV, hepatitis, syphilis and malaria from a single bloodsample without the need for intermediate sample treatment steps.

Utilising the present invention allows the assaying of small volumes ofwhole blood and thus the present invention provides a very rapiddiagnostic assay device that is simple to use and can be used in bothlaboratory and point-of-care field diagnostic locations. For example, afinger prick of blood would be sufficient to perform an assay. Similarlylarge volumes of sample can be used in this device by increasing theamount of absorbent material (second member). For instance, 10 mls ofdilute fluids like urine can be can be assayed to detect low abundancemolecules.

Analytes and/or ligands (e.g. antigens or antibodies) can be printeddown in titrating amounts and/or concentrations. Thus, in an individualscreen, this would provide a means of quantitating analyte-ligand levelswithin the sample solution.

BRIEF DESCRIPTION OF THE DRAWINGS

A specific embodiment of the present invention will now be described byway of example only and with reference to the accompanying drawings inwhich:

FIG. 1 is a schematic drawing of an apparatus embodying aspects of thepresent invention in a first configuration;

FIG. 2 is a schematic drawing of the apparatus of FIG. 1 in a secondconfiguration;

FIG. 3 is a perspective view of an assay apparatus or cassette embodyingaspects of the present invention;

FIG. 4 is an exploded view of the components of the cassette shown inFIG. 3;

FIGS. 5 a to 5 d show various stages in the use of the apparatus of FIG.3 in carrying out an assay; and

FIG. 6 is a graph comparing test results from samples spiked with alphaamalyse undergoing no-pre-incubation with samples undergoing a oneminute pre-incubation.

BRIEF DESCRIPTION OF A PREFERRED EMBODIMENT

Preparation of Detection Analyte

The entities bound to a gold colloid to produce the immunogold conjugate(normally immunoglobulins) are passively bound to give a stable complexthat retains the target activity of the antibody. The native goldcolloid dispersion as produced at Proteome Systems Limited (PSL) isinitiated with hydroxylamine reducing agent. Based on the assumptionthat the oxime is partially retained as a moiety in the colloid, theimmunogold conjugate is stabilised by addition of glutaraldehyde in thebinding step to form a Schiff's base between any residual oxime in thecolloid structure and free amines from the immunoglobulin.

The optimum binding profile for the immunoglobulin (concentration andpH) is obtained by titration of the antibody concentration against fixedaliquots of gold colloid at specific pH values and treatment of themixtures with saline solution. A sufficient concentration ratio forbinding between colloid and antibody prevents flocculation oraggregation of the colloid from suspension when treated with the salinesolution. Hence at low protein concentrations the addition of the salineresults in the colloid aggregating out of suspension, but when theoptimum level of antibody is reached the colloid remains stable insuspension (the integrity of the colloid suspension is measuredspectroscopically between wavelengths of 450 nm and 600 nm). This valueof antibody concentration gives the minimum protecting concentrationrequired to form the stable immunogold conjugate complex. An antibodyconcentration of 0.1 to 0.2 mg/mL in 5 mM borate is used in thetitration and for polyclonal antibodies a pH of 9 is chosen: this pH isnormally is well above the range of isoelectric point (pI) valuesencountered with the range of immunoglobulins in a polyclonal serum. Inthe case of monoclonal antibodies from a hybridoma the immunoglobulinshave a unique isoelectric point and the pH of the colloid is usually setat 0.5 to 1 unit above the pI of the antibody.

The concentration of antibody used for the conjugation is 110% of theminimum ‘protecting’ concentration determined from the titrationprocedure described herein.

Conjugation Protocol (for Single Antibody)

The required volume of colloid is measured out (assuming a 90% yield thevolume is nearly equivalent to that of the final conjugate at theoptical density measured for the colloid).

A volume of 5% glutaraidehyde solution is added to the rapidly-stirredcolloid to give a final glutaraldehyde concentration of 0.002%.

Five minutes after addition of the glutaraldehyde the calculated amountof antibody solution is added to the rapidly-stirred suspension. If asignificant volume of antibody is to be added (>10 mL) it is added in asteady stream of drops (preferably through a dropping funnel).

After 30 to 90 minutes depending upon the volume of colloid (from 200 mLto 5 L) the pH of the suspension is taken down from pH 9 to pH 7 (unlessthe conjugation is carried out between pH 7.5 to 6) with 0.2M phosphoricacid. A calculated volume of 10% (w/v) bovine serum albumin is added tothe suspension to give a final concentration of bovine serum albumin inthe suspension of 0.5%.

The suspension is left stirring for 3 to 4 hours or overnight when thevolume is >1 L and then centrifuged at 10,000 rpm for between 35 minutesand 60 minutes (depending upon volume and colloid size).

The supernatant is removed from the centrifuged suspension and theconcentrated liquid centrifugate taken up in 2 mM borate at pH 7.2containing 0.2% bovine serum albumin and 0.1% sodium azide (aspreservative).

Conjugation Protocol (for Multiple Antibodies)

For more than one antibody bound to the colloid, the protectingconcentration titrations (probed with saline for aggregation) arecarried out for each of the antibodies. The volumes of antibodies usedare those found for the individual antibodies (+10%) from thetitrations.

If equivalent binding levels of the antibodies at their individualoptimum binding levels are required, the antibodies are pre-mixed andadded together to the colloid (after glutaraldehyde addition). Theprocessing follows the procedure then described above for singleconjugations. If the antibodies are required to be at a particular ratioto each the primary antibody is added first and the secondary antibodiesadded at intervals later. The time course of the binding levels to thecolloid have been determined for some combinations of antibodies and thelevels of the second and subsequent antibodies decrease in a regularpattern with the time interval between addition of the first antibodyand subsequent additions. After 30 to 40 minutes interval a constantlevel of the second antibody bound at approximately 10% of the primaryreactant level is found. In some cases an interval of 5 minutes betweentest antibody and procedural control antibody at the requiredconcentration ratios is found to be optimal.

In one example, the primary antibody is mouse anti α amylase conjugatedat pH 8.3, and the secondary antibody (control) is goat immunoglobulinG. In another example, two primary antibodies (eg. anti-Human IgG1 andanti-Human IgG2) can be added simultaneously and a control antibodyadded at a suitable interval afterwards.

Preparation of Membrane with Capture Analyte

Capture analytes in the form of ligands such as antigens or antibodies(e.g. TB, HIV-1) are printed onto a protein-capture membrane matrix(e.g. a nitrocellulose membrane) in an appropriately sized array usingpiezoelectric chemical printing technology. A suitable chemical printingsystem for use in the present invention involves the use ofpiezoelectric drop-on-demand ink jet printing technology formicro-dispensing fluids in DNA diagnostics or the Combion Inc synthesisprocess called “CHEM-JET”. To explore drop on demand fluid dispensingfor DNA diagnostics, an eight fluid printer has been developed as partof the Genosensor Technology Development (GTD) project funded by theInstitute of Standards and Technology (USA). Research to date, isfocused on printing oligonucleotide micro-spots onto solid supports. Inthe CHEM-JET technique, which was developed at the California Instituteof Technology, tiny volumes of reagent bearing liquid are squirted ontospecific spots or addresses of a solid substrate much as an ink-jetprinter squirts ink onto a page. By repeatedly returning to each addresswith one or another of a small set of building blocks, in this case,nucleotides modified for the process, huge two-dimensional libraries ofshort DNA chains (oligonucleotides) can be assembled. Such a deviceincluding an imaging means is described in the applicant's co-pendingInternational patent application No. PCT/AU98/00265, the entire contentsof which are incorporated herein by reference. In the describedembodiment, antigen is printed onto a reaction membrane in 100 pldroplets, or multiples thereof (eg. 10 nl), with each aliquot being 1 mmapart. However, these volumes and distances can be increased/decreasedaccordingly depending on the chosen antigen titre and array size. Forexample, it is possible to dispense droplets with volumes as low as 1-10pl.

In a particularly preferred embodiment, antigens or antibodies can beprinted down in a matrix of dots or lines or in the shape of letters sothat quantitative multiple analyte analysis of a single sample ispossible.

After the dispensed antigen has dried, non-specific protein-bindingsites on the (nitrocellulose) membrane are blocked using 0.5% (v/v)casein in phosphate buffered saline (PBS) +0.05% (w/v) sodium azide+0.1% (v/v) Tween-20 (PBSA wash buffer). It is however an option toleave the membrane unblocked following the printing of the antigen (orantibody) or other ligand.

In another preferred embodiment syringe pump technology used for theadministration of liquids intravenously to patients can be adapted tolay down single or multiple lines on nitrocellulose membranes.

Use of Immunoconjugates in Flow-Through Format

The optical density of the immunoconjugate suspension is usuallymeasured at a wavelength of 520 nm. This value gives a relative measureof the concentration of the colloid. Also from knowledge of theconjugation conditions the level of antibody bound to the colloid at anoptical density=1 is known and this can be used to determine the optimumvolume to use in the flow-through test (antibody concentration per mL ofcolloid×volume of conjugate×optical density of conjugate).

The immunoconjugate is added to the sample that is pre-incubated in thefilter assembly prior to contact with the active membrane in thehousing. This enables the maximum level of binding of the analyte in thesample to bind to the active antibody conjugated to the colloid. Theconjugate: analyte complex is then captured by the immobilised testantibody on the membrane as the mixture filters through the activemembrane exposed to the sample fluid at the same time as the proceduralcontrol antigen binds to the secondary antibody linked to the conjugate.

For multi-analyte samples the multiple antibodies on the conjugate(detectable element) would bind at the same level to the sample anlaytesas the conjugated antibodies would be at their optimum binding levels onthe colloid particle.

Description of the Figures

Turning to the drawings, FIG. 1 shows a flow-through assay device 10,which utilises the nitrocellulose membrane described above. The deviceis in the form of a cassette 12 and an associated removable filter frame14. Inside the cassette there is the membrane (typically nitrocellulose)16 on which capture analytes in the form of ligands are printed, asdescribed above, which is located on top of an absorbent matrix 18. Theabsorbent matrix preferably comprises multiple layers of absorbenttissue or an absorbent pad such as blotting paper, in the specificembodiment twenty-four layers (double ply), which have been found topossess an ideal porosity that permits the most rapid flow-through ofvarious solutions. This rapid flow-through is important as it results inlower backgrounds with higher reaction specificity and higher signalresolution.

As shown in FIG. 1 the top of the cassette defines an opening in itsupper face and a depending generally frusto-conical well whose sidesdepend down as far as the membrane 16, to define a chamber havingsloping sides and a base defined by the membrane 16.

The filter unit frame 14 is spaced above the upper surface of thecassette 12. It also defines a depending conical well in the form of achamber 21 also referred to as a “pre-incubation chamber” having slopingsides and a base 22 formed from a 5 μm Whatman grade 1 membrane or a0.22 μm hydrophilic Durapore membrane filter (Millipore, North Ryde,Australia). However, other types of filter/membrane and pore size wouldbe suitable depending on the application. The function of the membraneis to retain a sample to be assayed in the well or pre-incubationchamber 21 long enough for a “pre-incubation step” to take place. Whenmembrane 22 is lowered to contact the membrane 16, capillary attractiondraws the sample from the chamber 20 through membranes 22 and 16 andinto the tissue 18.

For ease of use, two pins 24 are provided which support the filter frame14 at an appropriate distance above the cassette 12 during thepre-incubation step but which allow the filter frame to be pushed downso that the membranes 22 and 16 are in contact for the second stage ofthe process shown in FIG. 2. The frame 14 is also removable so that themembrane 16 can be viewed to determine the results of the assay.

FIGS. 3 to 5 d illustrate one commercial assay device design embodyingthe aspects of FIGS. 1 and 2.

In those Figures, the components which are equivalent to componentsshown in FIGS. 1 and 2 carry the same reference numerals. The cassette12 comprises an upper moulding 12 a and a lower moulding 12 b. Theporous membrane 22 is defined by the base of a pressed filter paperfrustro cone 22 a held in place by a filter retainer 23. The filter unitframe 14 defines two dimples 14 a on which an operator's thumbs maypress when depressing the filter frame to contact the membranes 22 and16.

FIGS. 5 a to 5 d illustrate the stages of operation of the apparatus.FIG. 5 a illustrates the filter frame separate from the cassette 12.FIG. 5 b illustrates the pre-incubation positioned with the base of thechamber/well 21 spaced from membrane 16. FIGS. 5 c and 5 d illustratethe device after the filter unit has been pressed down to bring themembranes 22 and 16 into contact to allow the sample to flow through tothe blottng paper 18.

If the membrane 22 is replaced with a hydrophobic membrane, it ispossible to operate the device with a pre-incubation step solely in theposition shown in FIGS. 3 and 4 with the membranes 22 and 16 always incontact. The hydrophobic membrane 22 will prevent flow of the sample inthe incubation chamber 21 to the reaction membrane 16. After asufficient period of time has past for detection analyte in the chamber21 to bind to the reagent, a suitable wetting agent is added to thesample in the chamber which allows the sample to flow through thehydrophobic membrane past the reaction membrane 16 and into an absorbentmatrix 20.

EXAMPLE 1

Application of the Pre-Filter Chamber

Whatman membrane (paper) or Reemay filters (polyester; 1 cm²) areinserted into the chamber 21 in the filter frame to form a conicalretaining vessel (pre-filter unit).

The sample is pipetted into the plastic pre-filter chamber (50-100 ul)along with a detection analyte in the form of a detecting antibody(50-100 ul) bound to colloidal gold (particle size 20-50 nm). The sampleis pre-incubated with the gold-conjugate (O.D.4) within thepre-incubation chamber for thirty seconds after gentle pippetting toensure adequate mixing. After thirty seconds the chamber is pressed intothe well 20 of the test cassette 12. Upon contact with the membrane 16containing the detection zone, the solution filters through to theabsorbent layer 18 beneath. The pre-filter 14 is discarded when thesolution has filtered through and two drops of PBSA wash buffer are thenadded to the reaction membrane to wash away excess gold-conjugaterevealing the results of the assay on membrane 16.

The use of the pre-incubation of the sample with the detection analyteincreases sensitivity by approximately ten fold. Further, anyparticulate matter is retained in the pre-incubation chamber all ofwhich can be removed to provide a clear signal. The use of thepreincubation chamber with the dual roles of permitting a pre-incubationstep and a pre-filtering step, also allows multi-analyte detection onthe reaction membrane by pre-incubating with a multi-analyte probe, e.g.colloidal gold bound to different detecting analytes. In addition,interfering analytes or substances that could cause false positives ornegatives in the assay can be removed or absorbed out in thepre-incubation step, e.g. human antibodies to mouse antigens can beabsorbed out by anti-HAMA antibodies.

Although the above described example relates to the antigens relating todisease, the immunoassay apparatus could be used, for example, as anallergy test kit, as a test kit for drugs of abuse or for analysingnon-human derived samples e.g. bovine, porcine, veterinary tests, andtests in agriculture such as grain quality evaluation, etc.

The method and apparatus of the present invention is particularly suitedto use with swabs which can be simply placed into the chamber 21,swirled around in liquid containing a detecting antibody (50-100 ul)bound to colloidal gold for 30 seconds before the pre-filter unit isdepressed to contact the membranes 22 and 16 together.

Any combination of ligands and analytes can be applied to the system ofthe present invention. The choice of ligands could be tailored to detectprevalent diseases in a particular country or population. For example,analytes from the following combination of diseases could be used fordiagnosis using this array.

-   -   1. TB and HIV    -   2. Hepatitis-B & C, HIV    -   3. Chagas, HIV, TB, Syphilis and Hepatitis-B & C    -   4. Malaria, Dengue, TB, Chagas.

Alternatively antigens representing different varieties of wheat orother agricultural products could be printed on the reaction membraneenabling detection of multiple strains with a single test.

EXAMPLE 2

The assay device can also be used for detecting analytes in body fluidsother than blood such as plasma, sera, urine, saliva and sputum. In thissystem the sample can be retained in the pre-incubation chamber 22 byuse of a hydrophobic membrane such as Reemay or Hollingsworth and Vose7303 instead of the Whatman grade 1 membrane or a 0.22 μm hydrophilicDurapore membrane filter described above. The sample is mixed with thedetection analyte for the required pre-incubation period. To obtainefficient flow through capillary action to the absorbent layer 18 whenthe pre-incubation chamber 22 is lowered onto the cassette 12, one oftwo procedures can be followed:

-   -   1. The membrane 16 containing the capture analyte is prewet with        at least one drop of wash buffer containing 0.01 M phosphate,        0.15 M NaCl, 0.0% Azide, 0.5% Tween 20 or any wetting agent        containing a detergent;    -   2. The membrane 16 containing the capture analyte is blocked        with a hygroscopic solution such as sucrose, trehalose,        fructose, or alternatively, glycerol. This changes the        characteristics of the membrane 16 from a non-hygroscopic to a        hygroscopic membrane allowing the sample to flow through to the        absorbent layer 18 upon contact of the membrane at the base of        the pre-incubation chamber 22 with membrane 16.

EXAMPLE 3 (COMPARATIVE EXAMPLE)

Comparison of no pre-incubation and 1 minute pre-incubation of a samplespiked with alpha amylase in the above described format.

Procedure

A 6% solution of bovine sera albumin was spiked with 0.1 ng/ml, 0.5ng/ml, 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/ml and 1000 ng/mland applied to the above format according to the following procedure.

No-Preincubation

I. The pre-incubation chamber was pressed down so that the base of thechamber comes into contact with the first member containing the captureantibody against alpha amylase.

II. Sixty microlitres of 0.5% tween in saline was added to thepre-incubation chamber and allowed to filter through to the absorbentmaterial beneath the first membrane.

III. One hundred microliters of spiked alpha amylase sample was added tothe chamber and allowed to filter through to the absorbent materialbeneath the first membrane.

IV. Sixty microlitres of 0.5% tween in saline was added to thepre-incubation chamber and allowed to filter through to the absorbentmaterial beneath the first membrane.

V. Sixty microlitres of anti-alpha amylase antibody linked to colloidalgold (particle size 20-50 nm) was added to the pre-incubation chamberand allowed to filter through to the absorbent material beneath thefirst membrane.

VI. Sixty microlitres of 0.5% tween in saline was added to thepre-incubation chamber and allowed to filter through to the absorbentmaterial beneath the first membrane.

VII. The pre-incubation chamber was removed and the result on thereaction membrane scanned with a densitometer. Signal strength wasmeasured in pixel intensity.

One Minute Pre-Incubation

I. Sixty microliters of 0.5% tween in saline was added to first membraneand allowed to filter through to the absorbent material underneath.

II. The pre-incubation chamber was suspended over the first membrane sothat there was a space between the chamber and the membrane.

III. One hundred microliters of spiked alpha amylase sample and 60microliters of anti-alpha amylase antibody linked to colloidal gold(particle size 20-50 nm) were incubated in the pre-incubation chamberfor 1 minute.

IV. The chamber was lowered until it came in contact with the firstmembrane and the mixture of sample and antibody-gold conjugate allowedto filter through to the absorbent material.

V. Sixty microliters of 0.5% tween in saline was added to thepre-incubation chamber and allowed to filter through to the absorbentmaterial.

VI. The pre-incubation chamber was removed and the result on thereaction membrane was scanned with a densitometer. Signal strength wasmeasured in pixel intensity.

Each data point on the graph is the average of two experiments using theapparatus described above. The results show that pre-incubation of thesample with the detection analyte has a minimal detection limit definedin pixel density of around 500 pg/ml of alpha amylase. This is comparedto a minimum detection limit without the pre-incubation of about 50ng/ml and indicates the pre-cubation increases the sensitive by around10 fold.

EXAMPLE 4 (COMPARATIVE EXAMPLES)

Demonstration of increased sensitivity with increased pre-incubation ofthe sample with the detection analyte.

Samples of amylase diluted in 0.5% saline to 400 ng/mL were treated withimmunogold conjugate against amylase and aliquotted onto theflow-through format in different protocols as shown below.

A. The sample was added to the format (without a filter present) andallowed to filter through prior to adding conjugate, followed by analiquot of conjugate immediately the sample had passed through themembrane.

B. The sample was mixed in the correct proportions with gold conjugateand aliquotted immediately onto the flow-through format.

C. The sample was mixed as with protocol B but added to the flow throughformat after a 60 second interval.

The results presented in pixel intensity are shown in the tables below(for 2 experiments) Sample Control Sample Control Protocol peak peakarea area S/PC ratio A 82 286 657 2120 317 B 288 758 2062 5509 383 C 823949 5843 6765 884 A 89 516 588 3890 588 B 482 830 3736 6345 602 C 708829 4506 5822 792

Clearly there is a significant increase in the sample signal when theanalyte is preincubated with the conjugate probe, as distinct tosequential detection on the flow-through format. The difference indetection levels (for the 400 ng/mL sample) equated to between a7.5-fold to 10-fold increase in detectable amylase in the flow throughformat when the sample is preincubated separately to the detectingcapture antibody.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A method for assaying for the presence of at least one pre-determinedreagent in a liquid sample comprising the steps of: a) providing a firstporous membrane to which capture analytes for binding to the reagenthave been bound; b) placing a liquid sample to be assayed and amulti-detection analyte in a chamber having a base defined by a secondporous membrane; c) allowing a sufficient period of time to pass for themulti-detection analyte to bind to said at least one reagent, ifpresent; d) contacting the base of the chamber with the first porousmembrane; and e) causing the sample to flow through the membranes toallow the reagent to bind to the capture analyte carried on the firstmembrane.
 2. The method as claimed in claim 1 further comprising thesteps of: removing the chamber and washing the first membrane with abuffer prior to inspecting the membrane for the presence of themulti-detection analyte.
 3. The method as claimed in claim 1 furthercomprising the steps of: washing the first membrane with a buffer byaddition to the chamber before removal and inspection of the firstmembrane for the presence of the multi-detection analyte.
 4. A methodfor assaying for the presence of at least one pre-determined reagentusing an apparatus comprising a first member comprising a first, porous,reaction membrane to which is bound a capture analyte for binding to areagent to be detected, the member having an upper surface and a lowersurface; a second member being a body of absorbent material disposedbelow and touching the lower surface of the first member; a chamber forcontaining a liquid sample spaced above the first member said chamberhaving side walls, and a base defined by a second membrane; the chamberbeing capable of retaining a liquid sample for a predeterminedincubation period; and means for supporting the chamber above the firstmember in two positions, a first position in which the membrane isspaced a sufficient distance from the first member so as to not permitfluid transfer from the chamber to the body of absorbent material, and asecond position in which the membrane is in contact with the firstmember, such contact permitting fluid transfer from the chamber throughthe first and second membranes to the body of absorbent material; themethod comprising the steps of: a) placing a liquid sample to be assayedand a multi-detection analyte in the chamber, with the chamber disposedin the first position; b) allowing a sufficient period of time to passfor the multi-detection analyte to bind the reagent, if present; c)depressing the chamber to the second position to contact the base of thechamber with the first porous membrane; d) allowing the sample to flowthrough the first and second membranes to allow the reagent, if presentto bind to the capture analyte carried on the first membrane.
 5. Themethod as claimed in claim 4, wherein the second membrane defined at thebase of the chamber is hydrophobic and the step of allowing the sampleto flow through the first and second membranes includes the addition ofa wetting agent on the upper surface of the second membrane prior tomoving the chamber to the second position.
 6. A method for assaying forthe presence of at least one pre-determined reagent using an apparatuscomprising a first member comprising a first, porous, reaction membraneto which is bound a capture analyte for binding to a reagent to bedetected, the member having an upper surface and a lower surface; asecond member being a body of absorbent material disposed below andtouching the lower surface of the first member; a chamber for containinga liquid sample spaced above the first member said chamber having sidewalls, and a base being defined by a second, hydrophobic, membrane,having an upper and lower surface, the chamber being supported above thefirst member with the lower surface of the hydrophobic membrane incontact with the upper surface of the first member, the base beingcapable of retaining a liquid sample in the chamber for a predeterminedincubation period, the method comprising the steps of: a) placing asample to be assayed and a multi-detection analyte in the chamber; b)allowing a sufficient period of time to pass for the multi-detectionanalyte to bind to the reagent, if present; c) adding a wetting agent tothe chamber; d) allowing the sample to flow through the first and secondporous membranes to allow the reagent, if present to bind to the captureanalyte carried on the first membrane.
 7. The method as claimed in claim1 wherein the sample is whole blood wherein whole red blood cells areremoved in the chamber by the second membrane acting as a filter andplasma in the blood is allowed to flow-through to the first membrane. 8.The method as claimed in claim 1 wherein the sample contains particulatematerials selected from the group including grain extracts, cell ormicrobial extracts wherein particulate materials are removed in thechamber by the second membrane acting as a filter.
 9. The method asclaimed in claim 1 wherein the sample comprises body fluids such asplasma, sera, urine, saliva and sputum and or wherein the secondmembrane is a hydrophobic membrane.
 10. The method as claimed in claim 1including the step of removing or neutralising unwanted analytes in thesample to be testing that may interfere with the binding of the sampleanalyte with the capture analyte on the first membrane in the chamber.11. The method as claimed in claim 10 wherein antibodies or otheranalytes which bind to the unwanted analytes are bound to the walls orthe base of the chamber.
 12. The method as claimed in claim 1 whereinthe sample to be tested is carried on an absorbent surface such as aswab or the like and including the steps of swirling the swab in anextraction solution in the chamber.
 13. The method as claimed in claim 1wherein the multi-detection analyte comprises a detection compoundselected from the group comprising an antibody, ligand and antigen. 14.The method as claimed in claim 13, wherein the antibody is animmunoglobulin.
 15. The method as claimed in claim 13, wherein themulti-detection analyte comprises two or more antibodies.
 16. The methodas claimed in claim 13, wherein the multi-detection analyte comprisesthree or more antibodies.
 17. The method as claimed in claim 1 whereinat least one antibody is a control antibody.
 18. The method as claimedin claim 1 wherein the multi-detection analyte comprises a detectableelement selected from the group comprising colloidal gold particle,latex bead, coloured dye and colloidal carbon.
 19. The method as claimedin claim 18, wherein the detectable element is a colloidal goldparticle.
 20. The method as claimed in claim 18, wherein the detectableelement is a latex bead linked to a coloured dye.
 21. The method asclaimed in claim 18, wherein the detectable element is colloidal carbon.22. The method as claimed in claim 15, wherein the two or moreantibodies are conjugated to a colloidal gold particle.